DNA damage recognition in linear and supercoiled DNA

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Special proteins in our cells have evolved to maintain genome integrity by recognizing and repairing damage in DNA caused by environmental pollutants, UV-rays from the sun, or mismatches introduced when DNA is replicated. These proteins need to rapidly scan DNA and yet slow down at potential “trouble spots” to recognize damage to begin repair. This process is complicated by the fact that DNA inside cells is looped and supercoiled (unwound or overwound). This project aims to uncover how DNA looping and supercoiling impact the interactions of DNA repair proteins with damaged DNA. The findings and the technologies developed in these studies will open doors for understanding how other proteins, such as those that regulate gene expression, engage with supercoiled DNA. The project will have educational impacts by providing opportunities for diverse undergraduate and graduate students to take part in cross-disciplinary cutting-edge research; developing an interdisciplinary undergraduate program in biophysics; engaging with high school teachers to develop teaching modules at the interface between physics and biology; and hosting high school students in their laboratories in the summer. Public and outreach activities will include a weekly “Saturday Morning Science” to bring in high school students to talk about science. A molecular-level understanding of DNA damage recognition is lacking, in large part because measurements of protein-DNA conformational dynamics on micro-to-milliseconds timescales relevant for interrogation and recognition are difficult and, thus, rare. Several lines of evidence suggest that damage sensing proteins sense differences in local DNA fluctuations and deformability to distinguish damaged from undamaged sites. Characterizing DNA dynamics and flexibility, however, has been a major challenge in the field. Even rarer are studies on how DNA topology (looping and supercoiling) – conserved across the domains of life – influence these dynamics. Using innovative fluorescence approaches that include laser temperature-jump, fluorescence lifetime-based FRET, and fluorescence correlation spectroscopy, this project will build on previous studies in the PI’s laboratory that unveiled protein-DNA conformational dynamics en route to damage recognition by Rad4, yeast ortholog of xeroderma pigmentosum C protein in the nucleotide excision repair pathway. Studies for mismatch recognition by MutS protein in the mismatch repair pathway will measure DNA dynamics for different mismatches, visualize how MutS engages with the mismatched sites, and ascertain whether mismatch interrogation/recognition rates for different mismatches correlate with MutS-mediated repair efficiencies. DNA minicircles designed with controlled superhelicity will be used to investigate the effect of looping and supercoiling on DNA fluctuations and what impact that has on the thermodynamics and kinetics of damage recognition by Rad4/MutS. The exquisitely high sensitivity and temporal resolution of these studies will enable measurements of Rad4/MutS-mediated kinking, unwinding, nucleotide-flipping kinetics at mismatched sites to form the recognition complex, shed light on how these dynamics are altered when DNA is supercoiled, and provide answers to how intrinsic DNA fluctuations stall and communicate “distress signals” to damage sensing proteins.This project is co-funded by the Genetic Mechanisms and the Molecular Biophysics Programs in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.